In nature, multiple enzymes in one metabolic pathway often form enzyme complexes to effectively catalyze cascade reactions via intermediate channeling effect. Similarly, covalent coupling of multiple enzymes is considered a very promising strategy to enhance the enzymatic reaction efficiency for production of value-added chemicals (non-patent document 1).
Although genetic fusion and covalent coupling of enzymes using amine or thiol groups have been utilized to construct multiple-enzyme complexes (non-patent document 2), these techniques have poor control over the coupling site and crosslinking process. Coupling at multiple sites inevitably generates a mixture of enzyme conjugates with varying compositions (non-patent document 3). Furthermore, conjugation to the enzyme active site most likely compromises the catalytic activities. To enhance the control over the coupling site and enzyme-complex configuration, several elegant approaches have been developed such as co-immobilization on a DNA scaffold, residue-specific incorporation of a non-natural amino acid and enzyme-mediated conjugation (non-patent documents 1, 4 and 5). However, there were still some restrictions in choosing coupling sites.
The inventors of the present disclosure have found out that a multi-enzyme conjugate exhibiting improved catalytic efficiency over respective free enzymes can be prepared by site-specific incorporation of a clickable non-natural amino acid into the enzymes and two compatible click reactions and have completed the present disclosure.